Figure 9.4 White blood cells (wbc), a speroid cell (sc), detached head (dh), a shed droplet (sd), and distal midpiece reflexes (dmr) on an eosin‐nigrosin stained smear.

Prior to beginning the evaluation of a semen sample, clean slides should be warmed on a warming stage for several minutes to prevent any artifactual, cold‐related changes. To prepare a semen smear, place a 4‐ to 5‐mm drop of stain at one end of the slide, adjacent to the frosted end if applicable. Frosted slides are ideal for labeling, which will facilitate the filing of semen smears as part of the medical record. Stain should always be placed first to limit exposure of the sperm to the damaging effects of chilling and desiccation before fixing with stain. Place a slightly smaller drop of semen adjacent to the stain and immediately mix the two together. Lifting the slide off of the warm stage with one hand and mixing with the other hand is far less awkward then leaving the slide on a flat surface. Wooden applicator sticks (Applicators, Puritan Medical Products Company LLC, Guilford, ME, USA) work well for obtaining droplets of semen and mixing. Using the stir stick, spread the mixture down the length of the slide in a stopping and starting fashion which will effectively create thick and thin areas of stain (Figure 9.5). This will help to provide the ideal area to count approximately 10–20 sperm per microscope field once the slide is dry. Another way to prepare a smear is to first mix the semen and stain as described above then use a second microscope slide to make the smear, similar to making blood smear. A better result will be achieved if the second slide is pulled back slowly, not rapidly as is the recommended practice when preparing a blood smear. Smears should be dried as quickly as possible to prevent the occurrence of bowed tails, an artifactual occurrence due to the hypotonic nature of eosin‐nigrosin. This may be accomplished by returning the slide to the slide warmer and by blowing on the slide to speed drying.

Figure 9.5 Semen smear preparation.

      The Feulgen staining technique is a multistep procedure that begins with an air‐dried smear. The exposure of the dried sperm cells to hydrochloric acid exposes aldehyde groups in the DNA which then bind with the Schiff's reagent, resulting in magenta staining of the DNA [27]. Feulgen staining is an excellent way to augment eosin‐nigrosin stained smears, particularly when abnormal DNA condensation is suspected or to get a more accurate differential count of the number and type of nuclear vacuoles.

      Classification and Evaluation of Sperm Morphology

Differential counts of sperm morphology are done with oil immersion, bright field microscopy at 1000× magnification. Always use immersion oil labeled for microscopy. Other oils, e.g. mineral oil, have a different refractive index and may penetrate the objective and effectively ruin it. Blood cell counters relabeled for use as sperm cell counters are an invaluable tool for the practitioner who evaluates more than a few bulls each year. At the Western College of Veterinary Medicine we label keys by the location of the defect on the sperm as shown in Table 9.1.

Table 9.1 Layout of an example label for keys used by the Western College of Veterinary Medicine, Saskatoon.

Head

Midpiece

Principal piece

Detached (loose) abnormal

Detached (loose) normal

Proximal droplet

Acrosome (other)

Normal

An image of a labeled sperm cell counter is shown in Figure 9.6. “Detached” or “loose” refers to detached heads. Detached heads with abnormalities, for example, the pyriform shape or nuclear vacuoles, should be counted as “detached abnormal.” Heads without abnormalities are counted as “detached normal.” Every sperm within a field should be counted to avoid bias. Exceptions will be if clumps of sperm are present obscuring other sperm, or if there are simply too many sperm preventing the complete inspection of individual cells. Fields should be inspected in systemic fashion – left to right, top to bottom has been an effective method for the author.

Figure 9.6 Cell counter with keys labeled for sperm cell morphology.

      Differential counts are reported as “defects per 100 cells.” Each time a key is pushed a cell is counted and added to the total. In cases where a sperm cell has more than one defect the respective keys should be depressed simultaneously. The two defects are counted, yet only one cell is added to the total. When the counter reaches 100 cells a bell is sounded. Counting just 100 sperm cells will be sufficiently representative if just a few abnormalities are recorded. When many abnormalities are encountered it is advised to count at least 300 cells to improve the reliability of the morphology assessment. Reliable counts should not differ by more than 10%, or, in other words, 10 cells. When reviewing my counts, I generally disregard any outliers in favor of completing another 100‐cell count. Once satisfied that my counts are representative I will report the average of the suitable counts.

      When sperm morphology is examined, several questions may arise about the specific type of defects observed:

       Is it an aberration or a significant abnormality?

       What would be the effect on fertilizing ability (sperm transport, binding to the oocyte, oocyte penetration, zygote formation)?

       What would be the tolerable level for bulls in natural service, or semen used in AI?

       What are the implications for the bull (cause, prognosis)?

      Readers of this chapter are encouraged to review sperm structure and spermatogenesis to develop a more complete understanding of sperm morphology (see Chapter 3). Classification systems have been developed to try to simplify semen evaluation; however, there appears to be quite a lot of misunderstanding of them. The earliest system used was the primary/secondary sperm defect system. By definition, a primary defect is one that originates during spermatogenesis – within the testicle – and a secondary defect is one that originates within the epididymis [28]. All head defects,

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